502. COLD SHOCK DURING RAPID COOLING OF SPERM FROM A TROPICAL ANURAN, THE CANE TOAD, BUFO MARINUS

Abstract

Amphibian sperm are not considered to be susceptible to cold shock injury during cooling and cryopreservation. In this study we investigated the susceptibility of the tropical bufonid, the Cane Toad (Bufo marinus) to sperm cold shock. Sperm from testes macerated in 2x Simplified Amphibian Ringer (inactivated state) were diluted 1:6 in various cryodiluents containing 10% sucrose and 10–20% glycerol or DMSO, or were used directly as undiluted controls. Samples were cooled at three cooling rates (1^oC min^–1, 5^oC min^–1 or placed directly on ice – rapid cooling) to 0^oC and then either warmed to room temperature and their motility and viability assessed after activation by dilution, or cryopreserved. Cryopreserved samples were stored in liquid nitrogen for two days and thawed at room temperature before assessment of motility and sperm viability. Cooling rapidly to 0^oC by directly placing samples on ice or cooling at 5^oC min^–1 before warming to room temperature resulted in a significant decline in motility (all means less than 40% of control motility after 30 min at room temperature) in comparison to samples cooled slowly at 1^oC min^–1 (all means greater than 80% motility; p < 0.05 to 0.01). Samples cryopreserved after cold shock (rapid cooling to 2^oC by immediate exposure of straws to 2^oC ambient temperature) versus samples cooled slowly (1^oC min^–1) to 2^oC prior to cryopreservation had significantly lower mean post-thaw motilities (p<0.05; in the range of 40–60% motile versus 80–95% for non-cold shocked). These data together indicate that the sperm of B. marinus undergo cold shock injury prior to freezing, and that post-thaw recovery after cryopreservation of cold-shocked sperm is substantially reduced.